US5135720A - Reaction chamber for amino-terminal sequence analysis of proteins or peptides - Google Patents

Abstract

A reaction chamber utilizes a sample carrier composed of a magnetic core and a surface coating effective to support sample of protein or peptide. The sample carrier is floated magnetically by means of electromagnets positioned within a reaction vessel. Edman reagnet is applied to the sample to effect amino acid sequence analysis of protein or peptide from amino-terminal. By such construction, reaction efficiency of repeated production of thiazolinon amino-acid derivatives is increased so as to increase number of identified amino acids, thereby enabling microanalysis of sample.

Description

BACKGROUND OF THE INVENTION

The present invention relates to a reaction chamber carrying out reactions which sequentially produce 2-anilino-5-thiazolinon amino acid derivatives in an analyzer which automates amino-terminal sequence analysis of protein or peptide.

FIGS. 5 and 6 show two kinds of the conventional reaction chamber carrying out reactions which sequentially produce thiazolinon amino acid derivatives based on the Edman reaction.

The conventional reaction chamber of FIG. 5 is constructed such that sample is adsorped in aglass filter 20 on amembrane filter 19 sandwiched by a pair ofglass blocks 18a and 18b within aframe 17, and reagent or solvent is applied to the sample through a flow path in the center of the glass blocks according to procedure of the Edman reaction.

The other conventional reaction chamber shown in FIG. 6 comprises areaction chamber 24 connected to avacuum pump 22 and to anitrogen gas bottle 23 through a three-way switch valve 21 to vacuum the reaction chamber or to fill the reaction chamber with nitroqen gas. Thereaction chamber 24 contains aglass cup 26 rotatable by amotor 25, asupply line 27 for delivering reagent and solvent needed for reaction, into theglass cup 26 and adischarge line 28 for removing this reagent and solvent.

However, with regard to the FIG. 5 conventional reaction chamber, the sample is supported between glass fibers of theglass filter 20 and therefore the reagent or solvent cannot be efficiently distributed to the sample. Hence the efficiency of reactions become lower. Thereby, repetitive yield in the sequence analysis is reduced. Such tendency becomes remarkable in the case of treating a micro amount of sample to thereby make unable the analysis. Further, various kinds of reagents and solvents are supplied through a common flow path to the protein sample for the reaction. Therefore, these reagents may be contaminated with each other.

With regard to the FIG. 6 conventional reaction chamber, the structure for rotation must be equipped in the vacuum chamber. Thereby, the maintenance of the analyzer is complicated. Especially, when the glass cup has a small dimension for treating a micro amount of the sample, it is difficult to maintain the stable rotation of such small glass cup.

SUMMARY OF THE INVENTION

An object of the present invention is to, therefore, eliminate the above noted drawbacks of the prior art.

According to the present invention, the reaction chamber is comprised of a reaction vessel made of nonmagnetic material having a reaction space and inlet and outlet of fluid such as solvent, a sample carrier disposed in the reaction space and comprised of a magnetic material and a sample supporting material covering the magnetic material, magnetic means disposed inside the reaction chamber for floating and holding the sample carrier by magnetic force to shift the sample carrier in a vertical axis direction, and a sensor for detecting a position of the sample carrier.

In such reaction chamber having the above construction, the sample of protein or peptide is uniformly distributed on the surface of the sample carrier in the reaction space, and the sample carrier is floated and held to shift in the vertical axis direction so as to increase the reaction efficiency between the sample and the reagent or solvent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 show embodiments of the reaction chamber according to the present invention, wherein

FIG. 1 is a sectional view of the reaction chamber,

FIG. 2 is a control block diagram ofelectromagnets 4 andposition sensors 5,

FIG. 3 is a sectional view of a sample carrier;

FIG. 4 shows a separation pattern of a standard mixture of phenylthiocarbamyl amino acid derivatives; and

FIGS. 5 and 6 are sectional views of the conventional reaction chamber.

DETAILED DESCRIPTION OF EMBODIMENTS

Hereinafter, embodiments of the present invention will be described in conjunction with the drawings.

Firstly, one embodiment is described to show how to float and hold a sample carrier in a reaction chamber. In thereaction chamber 1 shown in FIG. 1, areaction vessel 2 is provided therein with asample carrier 3 which carries a protein sample and is composed of magnetic material and is floated inside thevessel 2. This floating and holding is effected by magnetic force generated by electromagnets 4 (magnetic means) and the position of the sample carrier is monitored byposition sensors 5.

Theelectromagnets 4 are disposed within the walls of thereaction vessel 2 which is divided into upper and lower parts which are coupled to define the reaction space 6 to contain therein thesample carrier 3.

Further, thereaction vessel 2 is provided with anupper fluid path 7 and alower fluid path 8 so as to charge and discharge reagent and solvent needed for the reactions. Thereaction vessel 2 is supported by aretainer 9.

Next, the description is given with reference to FIG. 2 for how to control the floating and holding of thesample carrier 3 in the vertical axis by means of theelectromagnets 4 andposition sensors 5. Firstly, aposition sensor 5a detects a distance G₁ between anelectromagnet 4a and thesample carrier 3 floated by magnetic force generated by theelectromagnet 4a and anotherelectromagnet 4b, and anothersensor 5b detects a distance G₂ between theelectromagnet 4b and thesample carrier 3. In order to hold thesample carrier 3 at a mid point between theelectromagnets 4a and 4b, abridge circuit 10 processes a pair of detection signals representative of the detected distances G₁ and G₂, and then acomparator 11 compares the processed signal with a reference signal from areference signal source 12, and further asignal processing circuit 13 calculates appropriate values of electric currents for theelectromagnets 4a and 4b based on the compared results. Eachamplifier 14 amplifies electric currents to the electromagnets according to the calculated values so as to control the magnitude of the magnetic forces generated from theelectromagnets 4a and 4b to thereby equalize the distances G₁ and G₂ with each other. Further, when thesample carrier 3 is to be displaced upward or downward in the vertical direction, thesignal processing circuit 13 operates to calculate appropriate current values effective to enable theelectromagnetes 4a and 4b to adjust the distances G₁ and G₂ through theamplifiers 14.

In the inventive reaction chamber, applied reagents and solvents can be efficiently acted to the sample on the sample carrier.

Next, the description is given for how to sequentially produce thiazolinon amino acid derivatives from protein sample carried on the sample carrier and how to detect the derivatives.

As shown in FIG. 3, thesample carrier 3 is comprised of aspherical ferrite core 15 and aglass coating 16 formed thereon as the sample supporting material. In such structure, theferrite core 15 may be of spherical, cubic, cylindrical, spheroidic or other shape.

The sample supporting material coated on the surface of theferrite core 15 may be composed of glass, ceramics and polymer material such as polyvinylidenedifluoride and polymethyltrifluoropropylsiloxane.

The following procedure is based on an ordinary automated gas-phase Edman method. Thesample carrier 3 of 5 mm diameter is treated with polybrene (hexadimethrin bromide) and then is applied with 5 μl of 70% formic acid containing 1 pico (pico:10^-12) mole of myoglobin, and thereafter protein sample is dried. Subsequently, application of coupling reagent, buffer vapor, washing solvent, cleavage reagent and extraction solvent is delivered to the reaction chamber according to analysis program (Table 1) of the commercially available automated gas-phase sequence analyzer. Sequentially obtained thiazolinon amino acid derivatives are detected according to fluorescence analysis using 4-amino fluorescein. Namely, 75 μl of methanol containing 1% of pyridine and 25 μl of methanol containing 30 pico mole of 4-amino fluorescein are successively added to 150 μl of butyl chloride containing thiazolinon amino acid derivatives, and the mixture is dried. Next, 25 μl of methanol containing 30 pico mole of 4-amino fluorescein is added again, and the mixture is dried after 10 minutes of standing. This dried sample is dissolved by 50 μl of methanol. A 25 μl portion of the solution is applied to analysis using liquid chromatograph and fluorophotometric detector. Analysis condition is shown in table 2. Further, FIG. 4 shows separation pattern of the standard mixture of 20 kinds of phenylthiocarbamyl amino acid derivatives obtained by the above described procedure. An amount of the respective derivatives is in the order of 10 to 15 femto (femto:10^-15) mole. All of the derivatives can be separated and identified.

              TABLE 1                                                     ______________________________________                                    Cycle length: 32 steps                                                    Runtime: 43 mins 32 secs                                                  Step    Function       Value      Elapsed Time                            ______________________________________                                    1       Prep R2        6       0 min  6sec                               2       Deliver R2     20      0min  26sec                              3       Prep R1        6       0 min  32sec                              4       Deliver R1     2       0 min  34sec                              5       Argon Dry      40      1min  14 sec                              6       Deliver R2     400     7 min  54sec                              7       Prep R1        6       8min  0sec                               8       Deliver R1     2       8min  2sec                               9       Argon Dry      40      8 min  42sec                              10      Deliver R2     400     15min 22sec                              11      Prep R1        6       15min 28sec                              12      Deliver R1     2       15min 30sec                              13      Argon Dry      40      16min 10sec                              14      Deliver R2     400     22 min 50sec                              15      Argon Dry      120     24 min 50sec                              16      Deliver S1     60      25 min 50sec                              17      Deliver S2     200     29min 10 sec                              18      Argon Dry      120     31min 10sec                              19      Load R3        4       31min 14sec                              20      Argon Dry      4       31 min 18sec                              21      Pause          300     36 min 18sec                              22      Load S2        6       36min 24sec                              23      Block Flush    6       36min 30sec                              24      Argon Dry      120     38min 30sec                              25Prep Transfer  30      29min 0sec                               26      Deliver S1     9       39min 9sec                               27      Transfer w/S3  52      40min 1sec                               28Pause          20      40min 21 sec                              29      Transfer w/Argon                                                                         40      41min 1sec                               30End Transfer   1       41min 2 sec                               31      DeliverS3     30      41 min 32 sec                              32      Argon Dry      120     43 min 32 sec                              ______________________________________                                     (Extracted from 477 A type manual of Applied Biosystems Inc., Ltd.)       R1: 5% phenylisothiocyanate/heptane                                       R2: 12.5% trimethylamine/water                                            R3: trifluoroacetic acid                                                  S1: nheptane                                                              S2: ethyl acetate                                                         S3: butyl chloride

              TABLE 2                                                     ______________________________________                                    ANALYSIS CONDITIONS FOR LIQUID CHROMATOGAPH                               ______________________________________                                    Column:   Capcell Pack (AG) C18 produced by Shiseido                                co., Ltd. φ 4.6 mm × 150 mm                           column temperature: 43° C.                                         Detector: spectrofluorophotometer RT-540 produced                                   by Shimazu Seisakusho Co., Ltd.                                 Excitation wavelength: 494 mm                                             Emission wavelength: 513 mm                                               Pump: Waters 600E system                                                  Flow rate: total 0.8 m/min                                                Gradient program:                                                                       (A) 10 mM sodium phosphate buffer                                         (B) methanol                                                              (C) acetonitrile                                            ______________________________________                                    time (min) (A) %        (B) %   (C) %                                     ______________________________________                                    0.0        79           20      1                                         0.1        75           23      2                                         14.0       75           23      2                                         19.0       71           19      12                                        34.0       71           12      19                                        40.0       50           25      25                                        45.0       79           20      1                                         65.0       79           20      1                                         ______________________________________

As described above, in the reaction chamber according to the present invention, the sample carrier is floated and positioned in the reaction vessel, thereby reagents and solvents are efficiently and uniformly applied to the sample, as well as cross contamination of the used reagents and solvents can be avoided as much as possible.

Claims (12)

What is claimed is:

1. A reaction chamber comprising:

a reaction vessel composed of nonmagnetic material for defining a reaction space and having an inlet and outlet for charging and discharging fluid;

a sample carrier disposed in the reaction space;

magnetic means disposed inside the reaction vessel for generating magnetic forces effective to float the sample carrier into a desired position wherein the sample carrier is not in contact with the reaction vessel; and

a sensor for detecting the position of the sample carrier.

2. A reaction chamber according to claim 1; wherein the sample carrier is composed of a magnetic material and a sample supporting material formed on a surface of the sample carrier.

3. A reaction chamber according to claim 2; wherein the sample supporting material is composed of material selecting from glass, ceramics and polymers including poly vinylidene difluoride and polymethyltrifluoropropylsiloxane.

4. A reaction chamber according to claim 2; wherein the sample carrier has a spheric or spheroidic shape.

5. A reaction chamber according to claim 1; wherein the reaction vessel has a plurality of divided parts.

6. A reaction chamber according to claim 1; including means responsive to the detection of the position of the sample carrier by the sensor for controlling the magnetic means to maintain the desired position of the sample carrier.

7. A reaction chamber comprising:

a reaction vessel composed of nonmagnetic material and defining a reaction space having a fluid inlet and a fluid outlet;

a sample carrier disposable in the reaction space;

generating means for generating magnetic forces in the reaction space effective to float the sample carrier in the reaction space; and

means for sensing the position of the sample carrier in the reaction space and for controlling the generating means to maintain the sample carrier solely by magnetic forces in a desired position completely out of contact with the reaction vessel.

8. The reaction chamber according to claim 7, wherein the means for sensing comprises sensor for producing signals corresponding to the position of the sample carrier, and circuit means for processing the signals to produce contol signals for controlling the generating means.

9. The reaction chamber according to claim 7, wherein the sample carrier comprises magnetic material and has sample supporting material on a surface thereof.

10. A reaction chamber according to claim 9, wherein the sample supporting material is composed of material selecting from glass, ceramics and polymers including poly vinylidene difluoride and polymethyltrifluoropropylsiolxane.

11. The reaction chamber according to claim 7, wherein the sample carrier is spherical in shape.

12. The reaction chamber according to claim 7, wherein the reaction vessel comprises a plurality of connected parts.

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